5-(Thiophen-2-yl)isoxazoles as novel anti-breast cancer agents targeting ERα: synthesis, in vitro biological evaluation, in silico studies, and molecular dynamics simulation†

Herein, we report the design and synthesis of novel 5-(thiophen-2-yl)-4-(trifluoromethyl)isoxazoles (TTI), and in vitro evaluation of their anti-cancer activities. Based on the molecular structure of our previously developed isoxazole-based anti-breast cancer lead molecule, 3-(3,4-dimethoxyphenyl)-5-(thiophen-2-yl)-4-(trifluoromethyl)isoxazole (TTI-4), we designed a set of 14 new analogues of TTI-4. The TTIs are a synthetically challenging class of molecules, and we synthesized them with high purity by utilizing our in-house developed novel synthetic strategy, i.e., metal-free, cascade regio- and stereoselective trifluoromethyloximation, cyclization, and elimination strategy, with readily available α,β-unsaturated ketones by using commercially available and cheap reagents such as CF₃SO₂Na and ^tBuONO (cost-effective and sustainable synthesis). Subsequently, the anti-cancer activities of the newly synthesized molecules were evaluated against various cancer cell lines such as MCF-7, 4T1, and PC-3, and the molecules showed potential and more selective cytotoxicity against the human breast cancer cell line, MCF-7, among others. The in vitro screening revealed a new molecule, i.e., 5-(thiophen-2-yl)-4-(trifluoromethyl)-3-(3,4,5-trimethoxyphenyl)isoxazole (TTI-6), possessing an IC₅₀ value of 1.91 μM against MCF-7, is superior to the previous lead molecule (TTI-4) and also the best anti-cancer agent among all. The structure–activity relationship (SAR) studies revealed the importance of an unsubstituted thiophene ring in the 5th position, a –CF₃ functional group in the 4th position, and a highly electron-rich benzene ring bearing three –OCH₃ functional groups in the 3rd position of the isoxazole core to have superior activity. Further studies with TTI-6, such as apoptosis induction, cell cycle analysis, and nuclear staining, revealed an apoptotic cell death mechanism. The in silico molecular docking, induced fit analysis, and ADMET studies further supported the effects of various functional groups of TTIs on their anti-breast cancer activity by inhibiting the estrogen receptor alpha (ERα), a crucial nuclear hormone receptor involved in gene regulation that plays an important role in several human cancers.